Organisms encode multiple Hsp70s to increase capacity to regulate abundance of Hsp70 in accordance with need and to provide a range of distinct Hsp70 functions for carrying out specific tasks within all cells or in distinct cell types. We constructed a yeast system to evaluate Hsp70s from any source and are using it to investigate how Hsp70s within and across species influence propagation of amyloid in vivo and act in cellular protein quality control. The wide range of responses that prions have to alterations of Hsp70s and their co-chaperones provides a sensitive way to investigate even subtle functional distinctions among highly redundant Hsp70s and an approach to uncover the underlying mechanisms. We earlier identified a single Ala/Gly difference in the nearly identical Hsp70s Ssa1 and Ssa2 as solely responsible for differential ability of Ssa1 and Ssa2 to support propagation of URE3 prions. We extended this work to show this tiny difference also determined functional specificity of Ssa1 and Ssa2 in a cellular protein degradation pathway important for regulating abundance of gluconeogenic enzymes. Faulty regulation of these enzymes can cause diabetes. Our findings imply that regulation of Hsp70 substrate binding, not substrate binding per se, can underlie functional specificity. A large cohort of co-chaperones regulate and fine-tune Hsp70 substrate binding activity. Because intrinsic enzymatic activities of Ssa1 and Ssa2 are similar, we suspect that Hsp70 co-factors are responsible for the differences in how they function in protein degradation and we are working to identify them. J-proteins are obligate and highly abundant Hsp70 co-chaperones. Humans have a similar number of cytosolic Hsp70s as yeast, but over twice as many J-proteins. Thus, while intrinsic Hsp70 activities have diversified little, the ability of Hsp70 to perform additional or more complex tasks in humans has expanded, in part, by amplification of J-proteins. The extent to which J-proteins specify Hsp70 functions by providing their own unique substrate interactions or by their ability to recruit and regulate basic Hsp70 activities is far from understood. Another focus of our continued work is determining if differences in functions of Hsp70s are mediated by differences in the way they cooperate with J-proteins, other co-chaperones or other major chaperones. We are working to learn whether such differences in Hsp70 function contribute to protection from amyloid toxicity that we see in some of our strains and if human chaperones possess such protective functions. Another difference between Ssa1 and Ssa2 is that increasing expression of Ssa1, but not Ssa2, cures wild type cells of URE3. This difference is not determined by the Ala/Gly variation at position 83 that is responsible for the difference in the ways these Hsp70s support URE3 propagation. We are working to identify the structural and functional differences between Ssa1 and Ssa2 to determine how these Hsp70s differ in their anti-prion effects. Elevating Hsp70 can moderate pathology in models of protein folding disorders, while in the same models reducing Hsp70 activity can exacerbate, or alone even cause pathology. Hsp70 is therefore a promising therapeutic candidate for amyloid and other protein folding disorders and it is being studied as a drug target. Altering Hsp70 co-chaperones, in particular the J-proteins, also moderates pathology in several models of amyloid and other protein folding disorders. Our findings can help guide decisions about which Hsp70-family members would be most useful for such applications, or identify potential problems that could arise due to distinct sensitivities of different Hsp70s to specific compounds. Overall our work provides insight into functions of this chaperone system that can help guide strategies for using chaperones as targets for therapy in such diseases.